40

Angeli* S., Marchese S., Brandazza A., Petacchi* R. and Pelosi P.

Dip. Chimica e Biotecnologie Agrarie
via S. Michele, 4
56124 Pisa, Italy
ppelosi@agr.unipi.it

*Scuola Superiore "S. Anna"
56124 Pisa, Italy

Bacterial expression of a chemosensory protein (CSP) of Schistocerca gregaria

We have recently isolated from chemosensory organs of Schistocerca gregaria, several polypeptides of low molecular weight (around 14 kDa) and acidic nature. Their amino acid sequences revealed that all the isolated proteins belong to the same sub-family, whose first member was identified in Drosophila melanogaster and named OS-D. The five gene products, identified in both sexes, contain 109 amino acids, are very similar to each other and present four cysteine residues involved in two disulphide bridges, as demonstrated by mass spectrometry. This technique also confirmed the calculated molecular weights and excluded the presence of additional post-translational modifications.

Here we report the cloning of one of these genes into a pET-5 vector and the expression, purification and characterisation of the recombinant protein. The protein is expressed with an additional residue of methionine at the N-terminus, as the sole difference with the native polypeptide. Yields were in the range of 50-70 mg of soluble protein per litre of bacterial colture. Purification was accomplished by standard methods, involving ammonium sulphate fractionation, ion-exchange chromatography and gel filtration. The recombinant protein showed chemical properties identical with those of a native sample.

41

Marchese S., Brandazza A., Ceron S., Angeli* S., Minnocci* A., Mazzau, M. and Pelosi P.

Dip. Chimica e Biotecnologie Agrarie
via S. Michele, 4
56124 Pisa, Italy
ppelosi@agr.unipi.it

*Scuola Superiore "S. Anna"
uDip. dell'Ambiente e del Territorio, University of Pisa
56124 Pisa, Italy

Major chemosensory proteins of phasmids

The chemosensory organs of Phasmids express very high levels of soluble proteins of the class referred to as OS-D like or CSP (Chemosensory Proteins). Proteins of this class are also expressed in other orders of insects, such as Lepidoptera, Diptera, Imenoptera, Orthoptera and Blattoidea.

Here we describe the cloning and sequencing of some members of this class of proteins in the Phasmid species Eurycantha calcarata and Carausius morosus. Mature polypeptides are 100 to 117 residue long and contain four conserved cysteines. The same proteins or similar ones appear to be expressed in the antennae, in the tarsi and in other parts of the body. Polyclonal antibodies prepared against purified proteins from both species, as well as against a similar protein of S. gregaria, indicated no cross-reactivity, despite the sequence similarities between the three proteins. Each antiserum, however, stained bands of similar molecular weight in different chemosensory organs of the same species.

A comparison between all the known sequences of CSPs in different orders of insects is also presented and discussed.

42

Minnocci A., Angeli S., Monteforti G., Petacchi R.

BioLabs - SSSUP S. Anna
Via Carducci 40 I- 56127 Pisa (Italy)
microscopia@sssup1.sssup.it

Immunocytochemical distribution of a ChemoSensory Protein of Carausius morosus Br.

Isolation of soluble low molecular weight proteins in antennae, tarsi and labrum of S. gregaria, clearly showed the existence of a new class of Chemosensory protein (Angeli et al., 1999) likely to be involved in chemical communication. Similar proteins was also demonstrate to be present in Phasmids (Mameli et al., 1996), in particular in Carausius morosus (Tuccini et al., 1996). These proteins belong to a family, whose first member was identified in the antennae of D. melanogaster and named OS-D-like, described in different Orders of insects, from Phasmids to Lepidoptera and relatively well conserved across evolution. Polyclonal antibodies used to immunodetect these proteins in Schistocerca gregaria, showing that it is localized in contact chemosensory hairs of tarsi, antennae and labrum nor in olfactory sensilla.

We are currently investigating the ultrastructural localization of a CSP in the different sensory organs of this stick insect.

Angeli S., Ceron F., Scaloni A., Monteforti G., Minnocci A., Petacchi R. and Pelosi P. 1999. Structural characterisation, cloning and immunocytochemical localisation of Chemoreception proteins from Schistocerca gregaria. (in press)
Mameli M., Tuccini A., Mazza M., Petacchi R., Pelosi P., 1996. Soluble proteins in chemosensory organs of Phasmids. Insect Biochem. Mol. Biol. 26: 875-882.
Tuccini A., Maida R., Rovero P., Mazza M., Pelosi P.,1996. Putative odorant-binding proteins in antennae and legs of Carausius morosus. Insect Biochem. Mol. Biol. 26: 19-24.

43

Hartlieb Elke, Pröbstl Therese and Ziegelberger Gunde

Max-Planck-Institut für Verhaltensphysiologie
D-82319 Seewiesen
Germany
hartlieb@mpi-seewiesen.mpg.de

Odorant-binding proteins in the malaria mosquito Anopheles gambiae

Based on the increasing number of identified odorant-binding proteins (OBPs) within a given insect species together with their distinct distribution pattern among different sensilla and a few odor-binding studies, OBPs are believed to participate in odor discrimination.

We studied odorant-binding proteins in the haematophagous mosquito Anopheles gambiae and compared them with several hitherto identified OBPs of phytophagous insects. The heads including antennae, probosces and maxillary palps of tens of thousands of males or females of An. gambiae were used for preparative isoelectrofocusing. The fractions obtained differed in pH by appr. 0.05 units and were analysed in native PAGE, SDS-PAGE and immunoblots. According to the different protein pattern of males and females and the characteristically low molecular weight of OBPs (MWs below 20kDa) several soluble proteins with pIs between 4.1 and 5.2 were selected for N-terminal sequencing. All N-terminal sequences obtained, except for one, showed no homology to any other known sequence. Two sequences have been selected for peptide synthesis and antisera production.

In parallel, the fractionated proteins of An. gambiae have been checked for crossreactivity with five antisera raised against different OBPs of Lepidotera and four antisera against OBPs of Drosophila. Only with the polyclonal antisera against the native pheromone-binding protein of the silkmoth Antheraea polyphemus and to an even minor extent against the native general odorant-binding protein 2 of A. polyphemus, a weak crossreactivity was observed, indicating that An. gambiae OBPs might share only weak homology to Lepidoptera and Diptera OBPs.

Cross-section series of cryofixed terminal segments of male and female antennae of An. gambiae have been produced and investigated by transmission electron microscopy. Three main types of sensilla were found, two of them belonging to the category of single-walled wall pore sensilla (s. trichodea with olfactory function and long jagged s. chaetica with mechanosensitive function) and one being a double-walled type (grooved peg with olfactory function). The first An. gambiae OBP-antisera obtained are now used for immunocytochemistry to study the distribution of these proteins within the various types of odor-sensitive sensilla.

Supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR).

44

Krieger Jürgen, Picimbon Jean-Francois, Dietrich Karen and Breer Heinz

University Stuttgart-Hohenheim
Institute of Physiology
70593 Stuttgart
krieger@Uni-Hohenheim.de

Diversity of Odorant Binding proteins in Moth antennae

In moth and other insects odorants do not have direct access to the perceptive membranes of chemosensory neurons located in the antennal hair structure. On their way from the air towards specific receptors in the dendritic membrane of the olfactory receptor cell, pheromones and general odorants have to cross an aqueous fluid (sensillum lymph), which bathes and protects the dendrite, thereby representing a hydrophilic barrier for the hydrophobic odor molecules. The transfer of odor molecules through the sensillum lymph is supposed to be mediated by small, soluble binding proteins for pheromones and general odorants. Over the last decade odorant binding proteins from various insect species have been cloned and sequenced by several groups, indicating the existence of multiple binding protein classes. By means of molecular cloning approaches we have identified pheromone binding proteins (PBPs), two types of general odorant binding proteins (GOBP1s and GOBP2s) and ABPX proteins (antennal binding protein X) in the moth species Antheraea perny, Bombyx mori and Heliothis virescens. Recently, an additional type of antennal binding protein (OS-D) has been discovered in Drosophila. To evaluate if a related binding protein may also exist in moth antennae, we set out to search for OS-D homologues in Bombyx mori and Heliothis virescens. Application of PCR- based and differential screening approaches, led to the isolation of clones from antennal cDNA libraries of each species, which encode proteins with significant sequence identity to Drosophila OS-D, including 4 conserved cysteines at identical position along the polypeptide chain. Two different OSD-like proteins were identified in Bombyx (BmorOS-D1 and 2) and Heliothis (HvirOS-D1 and 2).

Northern blot analysis confirmed the expression of the OS-D homologues in the antennae; interestingly, OS-D expression was also detected in legs, in which chemosensory neurons are also found. Our results indicate that at least five different binding protein classes (PBP, GOBP1 and GOBP2, ABPX and OS-D) are expressed in moth antennae. The increasing number and diversity of putative odorant binding proteins in moth antennae, together with a different topographic distribution in sensilla with unique functional identity, suggests that each class of binding protein may be tuned to interact with a certain group of odorants, thus acting as a selective signal filter in the antennae of insects.

This work was supported by the Deutsche Forschungsgemeinschaft.

45

Shanbhag S., Müller B., Steinbrecht R.A.

Max-Planck-Institut fuer Verhaltensphysiologie
82319 Seewiesen, Germany
steinbrecht@mpi-seewiesen.mpg.de

The complex pattern of odorant-binding proteins in Bombyx and Drosophila

Molecular cloning approaches have allowed for the identification of an increasing number of odorant-binding proteins (OBPs) in insects. Thus, in the antenna of the silkmoth Bombyx mori meanwhile four OBPs have been characterized: PBP, GOBP1 and GOBP2 belong to the well-known families of lepidopteran pheromone-binding and general odorant-binding proteins, while ABPX represents a novel class with slight amino-acid homology to Drosophila OBPs 1. In Drosophila melanogaster, the number of putative odorant-binding proteins (OBPs) is even larger. Recombinant proteins were used to raise polyclonal antibodies against the four Bombyx OBPs and the following proteins of D. melanogaster: OS-E, OS-F, PBPRP2, PBPRP5, LUSH [collaboration with M. Mameli, J. Krieger, H. Breer (Stuttgart-Hohenheim); D. Hekmat-Scafe, J.R. Carlson (New Haven); C.W. Pikielny (Piskataway), D.P. Smith (Dallas)].

In a post-embedding labelling protocol we used these antisera on ultrathin sections of cryofixed Bombyx and Drosophila antennae in order to find out the localization of these OBPs at electron microscopic resolution. The resulting expression pattern in the different types of olfactory sensilla was complex but persistent.

Bombyx: Whereas PBP and GOBP2 were not co-localized in the same sensilla, as reported earlier, this is different for GOBP1 and ABPX. Long s. trichodea, which in male silkmoths express PBP, also may express GOBP1 and ABPX, albeit at a very low level. In the short s. trichodea and s. basiconica, however, several subtypes with different labelling characteristics could be discriminated. Some combinations were particularly common; e.g. many s. basiconica expressed both GOBP1 and GOBP2 at relatively high level. Medium-sized s. trichodea and intermediate sensilla were found in at least 5 specific labelling patterns with sex-specific frequency distribution.

Drosophila: OS-E and OS-F were always co-localized and were present in the great majority of sensilla trichodea; these proteins were also labelled in a subset of small s. basiconica. LUSH was found in the majority of s. trichodea too. Thus, this sensillum type expresses three different OBPs in the same sensory hairs. PBPRP5 was observed in a subset of large s. basiconica, while PBPRP2 was ex-pressed in a very small fraction of the s. coelo-conica on the antennal surface.

Thus, a highly complex expression pattern of OBPs is emerging in both species, with sensillar subtypes as defined by their labelling characteristics by far outnumbering the morphologically defined types. This possibly reflects the pattern of functional specificity of these sensilla.

46

Vogt Richard

Department of Biological Sciences
University of South Carolina
Columbia, SC 29208
vogt@biol.sc.edu

OBP Diversity and Insect Phylogeny: Implications on the Evolution of Insect Olfaction.

Cloning and expression of LAP, an Adult Specific Odorant Binding Protein of the True Bug Lygus lineolaris (Hemiptera, Heteroptera).
Insect Odorant Binding Proteins (OBPs) have been identified from numerous species representing several insect Orders including Lepidoptera, Diptera, Coleoptera and Hymenoptera. All of these are holometabolous insects belonging to a presumed monophyletic division of insects known as the Endopterygota. Recently, LAP, an antennal protein with OBP-like properties, was identified from Lygus lineolaris, a hemipteran insect representing the Hemipteroid Assemblage, a sister division to the Endopterygota. The full length sequence of LAP is presented in this report. In situ hybridization analysis reveals LAP mRNA expression in cell clusters associating with olfactory sensilla; expression is adult specific, although it initiates in developing adult tissue during the transitional period that precedes the actual adult molt. LAP sequence is shown to be significantly related to the OBP-related protein family, especially the OS-E and OS-F proteins of Drosophila (Diptera), the ABPX proteins of the Lepidoptera and the OBPRP proteins of the Coleoptera. Current models of OBP action under investigation suggest that OBPs are the first biochemical step in odor reception capable of some level of odor discrimination. Assuming that the OBP-related proteins represent one homologous family, the identification of LAP significantly expands knowledge of the phylogenetic depth of that family and its underlying role in odor detection to encompass all members of the Endopterygota and Hemipteroid Assemblage, which include many species of considerable agricultural and health-related importance.

47a

Kaissling Karl-Ernst

Max-Planck-Institut für Verhaltensphysiologie
82319 Seewiesen/Starnberg
Germany
kaissling@mpi-seewiesen.mpg.de

Model of perireceptor and receptor events in olfactory sensilla

A mathematical model of perireceptor and receptor events has been developed for olfactory sensilla on the antennae of the moth Antheraea polyphemus. The reaction network includes the adsorptive uptake of pheromone molecules by the olfactory hairs (see Fig. 1, reaction 1), the diffusion of the pheromone molecules pheromone from the hair surface to the receptor cell (Fig. 1A, dashed arrows), the formation of a complex of pheromone (F) and the pheromone binding protein (PBP), with its reduced form (Bred) (reaction 2), and its oxidized form (Box) (reaction 5), the interaction of this complex with receptor molecules (R) on the plasma membrane of the olfactory cell (reaction 3), the deactivation of the pheromone (reaction 4), and finally its enzymatic degradation (reactions 6 - 11). The model is consistent with morphometrical, radiometrical, electrophysiological and biochemical data reported by several authors (for references see Kaissling, 1998 a, b). It includes the following assumptions and hypotheses:

- the diffusion coefficient for the transport of pheromone from the hair surface to the receptor cell is equal to the one determined for longitudinal migration on the hair,
- only the complex FBred activates the receptor molecules,
- the observed redox shift FBred --> FBox deactivates the pheromone molecules,
- the redox shift is catalyzed by the receptor molecules,
- pheromone molecules bound to PBP are largely protected from enzymatic degradation, i. e. rate constants k8 and k10 are assumed to be 1000 fold smaller than k6,
- the dendritic membrane area is maximally covered with receptor molecules, and
- the duration of the elementary receptor potentials elicited by single pheromone molecules reflects the lifetime of the ternary complex FBredR.

On the assumption that the dose-dependent relationship between receptor occupation and potential amplitude during transients is the same as that measured for the equilibrium state, the model allows to simulate the time course of the receptor potential, including saturation effects at high stimulus intensities, and the minimum delay of the onset of the receptor potential at high stimulus intensities (Fig. 2). Together with the experimental data the simulation is used to determine tentative rate constants for the reactions involved in the model (Fig. 1B).

The simulation of the receptor potential kinetics suggests that the proposed deactivation mechanism (the redox shift) rather than enzymatic pheromone degradation, is responsible for the primary decline of the receptor potential after the end of stimulation. The main function of the degradation is to fully eliminate the pheromone molecules from the olfactory hairs for maintaining the high sensitivity of the receptor cell. In addition to the free F, also the complex FBox must be degraded (Fig. 1A, reactions 8, 9), although with a 1000 fold lower affinity to E, in order to explain the velocity of pheromone degradation measured in situ.

The study supports the idea that perireceptor and receptor events rather than intracellular signalling processes govern the kinetics of the receptor potential. This was suggested by characteristic differences observed in the kinetics at stimulation by certain pheromone derivatives. The primary decline of the receptor potential depended on the structure of the stimulus molecule which may be simulated by varying the rate constant k3.

Acknowledgement: The computer model was developed by J. Thorson, Oxford.

Kaissling, K.E. (1998a) Flux detectors versus concentration detectors: two types of chemoreceptors. Chemical Senses, 23, 99-111.
Kaissling, K.E. (1998b) Pheromone deactivation catalyzed by receptor molecules: a quantitative kinetic model. Chem. Senses, 23, 385-395

47b

Minor Alexander V.¹, Kaissling Karl-Ernst, Thorson John²

Max-Plank Institut für Verhaltensphysiologie, Seewiesen, 82319 Starnberg, Germany
kaissling@mpi-seewiesen.mpg.de
¹Institute of Ecology and Evolution, Moscow 117071, Russia
²Computational Epistemology, Combe, Oxford, U.K.

Elementary electrical events in olfactory cells of the silkmoth Bombyx mori

Elementary receptor potentials of pheromone-sensitive olfactory cells in moth antennae, which can be observed in response to low pheromone concentration, appear as discrete "bumps" of few ms duration or as bursts of such bumps (Kaissling and Thorson,1980; Kaissling 1994). A single bump reflects a transient depolarisation of a receptor cell which corresponds to an increase of the membrane conductance up to several tens of pS. One bump presumably results from the activation of one or several ion channels after the pheromone molecule is bound to a receptor molecule on the membrane of the receptor-cell dendrite. The durations of single bumps and their bursts look like random events and can vary in a wide range from a few ms to hundreds of ms.

In the present study elementary receptor potentials (ERPs) and elementary receptor currents (ERCs, under transepithelial voltage clamp) were recorded from open tips of pheromone-sensitive sensilla in the male moth Bombyx mori. The main aim of recording was measuring the durations of bursts, of single bumps within bursts and of intervals (gaps) between two adjacent bumps in the burst, to estimate the stochastic properties of the bump-generating mechanism. The time course of a single bump-like potential change recorded from the sensillum tip consists of two transients (rising and falling parts) with rather sharp onsets. We found that this time course could be successfully simulated by short step-like increases of membrane conductance in the outer segment of the cell dendrite. For the simulation we developed an equivalent electrical circuit of the sensillum which took into account both the membrane resistances and capacitances of the sensory and auxiliary cells. When the sensillum currents were recorded in the voltage-clamp mode the bumps had a more rectangular appearance, indicating that the influence of membrane capacitances was partially compensated.

Burst, bump and gap durations and the number of bumps within a burst were measured in the bombykal-sensitive receptor cells. It was found that the probability density functions (pdf) of bump duration (d2) and gap duration (d3) can be approximated by single exponentials with time constants of about 6 - 10 ms (D3) for the bumps and 40 - 50 ms (D2). for the gaps. The pdf of the burst durations (d4) (considering one-bump events also as bursts) is a combination of two exponentials in the form: pdf(t)= A1*Exp(t/D41) + A2*Exp(t/D42) where the shorter and the longer time constants are in the range of 10 - 20 ms and 150 - 200 ms, respectively.

References:
Kaissling K.E. and Thorson J. (1980) in: Receptors for Transmitters, Hormones and Pheromones in Insects, D.B. Sattelle, L.M. Hall and J.G. Hildebrand, eds. Elsevier, Amsterdam, 261-282.
Kaissling K.E. (1994) in: Int. Symp. Olfaction and Taste XI, K. Kurihara, N. Suzuki and H. Ogawa, eds., Springer, Tokyo, p 812-815.
Kaissling K.E. (1998). Chem. Senses 23:385-395

48

Redkozubov Alexei and Belousova Tatiana

Institute of Physiologically Active Substances
Russian Academy of Sciences
142432 Chernogolovka, Moscow region, Russia
redkozub@ipac.ac.ru

Cyclic GMP reduces the response of the moth's olfactory receptor neurone to pheromone

Odour reception is a result of complex biochemical and electrophysiological reactions which occur in olfactory receptor neurons (ORN). Second messengers as for instance cyclic nucleotide monophosphates are involved in the reactions. Cyclic nucleotide monophosphates contribute to an excitatory signal transduction pathway as well as to an inhibitory pathway and to adaptation processes in the ORN. Possible involvement of guanosine cyclic monophosphate in olfactory transduction in insect is suggested because the cyclic nucleotide and guanylate cyclase occur in moth antennae (Ziegelberger et al. 1990, Boekhoff et al. 1993). However the role of cyclic nucleotides in signal transduction in moth's ORN still remains to be elucidated since a number of provocative data concerning particularly this role were collected (Villet 1978, Ziegelberger et al. 1990, Zufall and Hatt 1991, Zufall et al. 1991). Here we report an inhibitory effect of the membrane permeable dibutyryl cyclic GMP on the response of the olfactory receptor cell of the moth Bombyx mori to its sex pheromone bombykol.

The effects of membrane permeable dibutyryl cyclic GMP (cGMP) on the bombykol-elicited receptor current and nerve impulse activity of the ORN were studied using the open sensillum recording technique. cGMP was applied to the outer dendritic membrane of the ORN. cGMP (0.1 mM) reduced by about 75% the amplitude and slowed down about ten times the rising kinetic of the overall receptor current activated by a pulse of strong pheromone stimuli. Thus an increasing cGMP level in the outer dendrite of the ORN reduces the amplitude and slows down the kinetic of the response of the receptor cell to pheromone such as if the stimulus intensity was smaller by several orders of magnitude. cGMP (0.1 mM) diminished by about 87% the nerve impulse frequency elicited by continuously applied weak pheromone stimuli. Similarly, the increased concentration of cGMP in the outer dendrite of the ORN reduces the bombykol-elicited nerve impulse activity to the level of the spontaneous impulse activity observed without pheromone.

The observed inhibition of the response to pheromone was due to size reduction by about 44% of the elementary receptor current which elicits the nerve impulses and underlies the overall receptor current.

The down-regulating effect of cGMP on the pheromone response of the ORN described here implies that an inhibiting transduction pathway, involving guanylate cyclase and cGMP, is probably present in the ORN of the moth. The fact, that response to strong pheromone stimuli becomes under cGMP as little and as slow as the response to weaker pheromone concentration suggests that cGMP is a factor which may adjust the cell sensitivity to odour and play a role in olfactory adaptation.

49

Marx Thomas¹ , Gisselmann Günter¹ , Störtkuhl Klemens F.² , Hovemann Bernhard T.² & Hatt Hanns¹

¹ Ruhr-Universität-Bochum
Fakultät für Biologie
Lehrstuhl für Zellphysiologie
44780 Bochum, Germany
Thomas.Marx-2@ruhr-uni-bochum.de

² Ruhr-Universität-Bochum
Fakultät für Chemie
AG Molekulare Zellbiochemie
44780 Bochum, Germany

A novel putative voltage- and cyclic nucleotide-gated ion channel from Drosophila melanogaster

The second messengers cyclic 3',5'-guanosine monophosphate (cGMP) and cyclic 3',5'-adenosine monophosphate (cAMP) play an important role in visual and olfactory signal transduction in both vertebrates and invertebrates. Cyclic nucleotides can act directly on cyclic nucleotide-gated (CNG) ion channels, which are responsible for unspecific cationic conductances in vertebrate olfactory and visual receptor cells (Kaupp, 1995). Recently, we were able to demonstrate the presence of cAMP-gated potassium channels in the outer dendritic membranes of lobster olfactory receptor neurons (Hatt and Ache, 1994).

Therefore we were particularly interested in newly discovered cDNAs, cloned from see urchin, mouse and human, the predicted polypeptides of which resemble CNG potassium channels (Santoro et al., 1997; Santoro et al., 1998; Ludwig et al., 1998; Gauss et al., 1998). The recombinantly expressed channels of this type show general electrophysiological properties of Ih channels, which are dually gated by hyperpolarization and cyclic nucleotides (Pape, 1996).

The amino acid sequence from the mouse Ih channel "BCNG-1"(Santoro et al., 1997) was used to design a set of PCR primers corresponding to putative conserved amino acids. The PCR product amplified from Drosophila melanogaster showed high homology to BCNG-1. We isolated a full length clone from a Drosophila melanogaster head cDNA library. The predicted polypeptide, named Drosophila melanogaster Ih channel (DMIH), consisted of 945 amino acids. DMIH shows significant homology to both the voltage-gated potassium channels and the cyclic nucleotide-gated channels. DMIH contains six putative membrane-spanning segments (S1-S6), where the S4 segment is likely to be a voltage sensor. Between S5 and S6 we found a pore forming region with a GYG motif, which is typical for potassium channels. The carboxy terminal region exhibits a cyclic nucleotide binding domain.

Reverse transcription-polymerase chain reaction experiments showed, that the channel mRNA is expressed in head and antennae of Drosophila. To localize the expression of DMIH mRNA we performed in-situ-hybridization with digoxygenin labeled antisense RNA of DMIH to cryosections of Drosophila heads. Expression was detected in the compound eye, the olfactory organ and the auditory organ.

The fact, that the DMIH mRNA is expressed in various sensory tissues, lead us to conclude, that this channel might play a distinct role in the general sensory signal transduction in Drosophila.

Preliminary electrophysiological data, obtained from transfected HEK293 cells, show a functional expression of DMIH.

References:
Gauss, R., Seifert, R. and Kaupp, U.B. (1998), Nature, 393, 583-587.
Hatt, H. and Ache, B.W. (1994), Proc.Natl.Acad.Sci.USA, 91, 6264-6268.
Kaupp, U.B. (1995), Curr.Opin.Neurobiol., 5, 434-442.
Ludwig, A., Zong, X.G., Jeglitsch, M., Hofmann, F. and Biel, M. (1998), Nature, 393, 587-591.
Pape, H.C. (1996), Annu.Rev.Physiol., 58, 299-327.
Santoro, B., Grant, S.G., Bartsch, D. and Kandel, E.R. (1997), Proc.Natl.Acad.Sci.U.S.A., 94, 14815-14820.
Santoro, B., Liu, D.T., Yao, H., Bartsch, D., Kandel, E.R., Siegelbaum, S.A. and Tibbs, G.R. (1998), Cell, 93, 717-729.

50

Stoertkuhl, K.F., Hovemann, B., and Carlson, J.R¹

Ruhr-Universität Bochum
44780 Bochum
FRG
klemens.stoertkuhl@ruhr-uni-bochum.de

¹ Department of Biology, Kline Biology Tower, Yale University
New Haven CT, 06520-8103, USA

Olfactory adaptation in wild type and the mutant TRP of Drosophila melanogaster

To examine adaptation of the olfactory system we used a modified T-maze. Flies which were pre-exposed to a high dose of isoamyl acetate (IAA) for 60 sec showed a reduced response to a repellent IAA stimulus in the T-maze. Their response index was 20% that of naive flies. Further investigation showed that adaptation occured within 30 sec of an adaptive stimulus and recovery occurred on the order of minutes. Cross-adaptation was also observed, in that flies pre-exposed to IAA showed a reduced response to a variety of other odorants in the T-Maze.

In contrast, the mutant transient receptor potential (trp), bearing a defect in visual system electrophysiology, showed no adaptation in the adaptive olfactory T-maze. The trp gene likely codes for a voltage-independent Ca2+ channel. Interestingly, the olfactory adaptation defect in trpp301 is dominant.

Olfactory adaptation was shown to occur in the periphery. In the wild-type, the amplitude of electroantennograms (EAGs) was shown to be smaller in pre-exposed animals than in naive animals. Although odorant stimulation of naive trp mutants elicits a normal EAG amplitude, trp mutants were shown to be abnormal in antennal adaptation. Surprisingly, whereas wild-type flies recovered to 50% of the naive response at 2-3 min, trpp301 mutants recovered faster, showing 50% of the naive response after 1.5 min.

Immunostaining with a polyclonal serum against Trp showed labeling of the third antennal segment at 70h APF but no staining in the mature antenna. Further investigation with temperature shift experiments confirmed that Trp is essential at 70h APF to show normal olfactory adaptation in the mature antenna.

51

Liscia A., Solari P. and Crnjar R.

Dipartimento di Biologia Sperimentale, Sezione di Fisiologia Generale,
Université di Cagliari,
Cittadella Universitaria SS 554 Km 4,5 Italy

Is salt and water reception in the blowfly mediated by the CA++ cascade?

On the basis of previous results, Liscia et al. (1997) reported that NaCl reception in the blowfly, unlike what observed in vertebrates, is not mediated by an amiloride- sensitive cation conductance.
With the present study we have investigated the effects of W-7 (a calmodulin antagonist involved in the Ca++ cascade; Hidaka et al., 1981) and of the Ca++ blockers EDTA and EGTA on the response of the "salt" and "water" cells of the labellar chemosensilla in the blowfly Protophormia terraenovae, to stimulation with NaCl or KCl.

Our electrophysiological data indicate that W-7 almost suppresses the "salt" chemoreceptor response to both salts. Besides, both EDTA or EGTA have an inhibitory effect on the stimulating effectiveness of the two salts tested. The fact that W-7 virtually blocks both NaCl and KCl stimulation on the "salt" cell points to the existence of a common Ca++-sensitive transduction mechanism for K+ and Na+ either involving Ca++-calmodulin complexes or direct W-7 effects at the membrane level such as blocking of Ca++ channels (Bergquist, 1989). On the other hand, the smaller diminishing effect of the two Ca++ blockers tested (EDTA and EGTA) on the responses to either salt supports the idea that the strong inhibiting action exerted by W-7 involves a different mechanism - either additional or alternate - possibly based upon Ca++-calmodulin complexes. The spike activity evoked by NaCl from the "water" unit appears to be enhanced by W-7 in a concentration-dependent manner, while is affected by neither EDTA nor EGTA. W-7 may have a stimulatory effectiveness by itself on the dendritic membrane of the "water" cell in the presence of NaCl or, if the "osmometer" hypothesis is to be credited instead, W-7 may act indirectly on the intracellular osmolarity of the chemoreceptor, by modulating the Ca++ binding affinity of calmodulin. In fact, the Ca++-calmodulin complex and/or the free intracellular Ca++ could control the cell volume by a yet unknown mechanism.

1. Bergquist B. L. (1989) Trans. Am. Microsc. Soc., 108 (4), 369-379.
2. Hidaka H., Sasaki Y., Tanaka T., Endo T., Ohno S., Fuji Y. and Nagata T. (1981) Proc. Natl. Acad. Sci. USA , 78, 4354--4357.
3. Liscia A., Solari P., Majone R., Tomassini Barbarossa I., Crnjar R. (1997) Physiol. Behav. 62, 875-879.

52

Lucas Philippe

INRA - Unite de Phytopharmacie
Route de Saint-Cyr
78026 Versailles CEDEX
plucas@versailles.inra.fr

Identificationof an Inwardly Rectifying Hyperpolarization-activated channel in cultured accessory cells of Mamestra brassicae

53

Meunier Nicolas, Ferveur Jean-Francois, Marion-Poll Frederic

INRA Station de Phytopharmacie, Etoile de Choisy
Route de Saint-Cyr
78026 Versailles CEDEX
nmeunier@versailles.inra.fr

First electrophysiological study of the leg taste receptor in Drosophila melanogaster

Drosophila leg taste receptors are involved in major behavioral events such as sexual interaction and food searching. Up to now, this sensory system has been characterized by a combination of behavioral, anatomical and genetical approaches.

We present for the first time electrophysiological evidences of their sensitivity. Taste sensilla on the legs can be grouped in three types of responses. The first group of sensilla responds tonically to sugar, salt and water; the second group responds phasically to salt and sugar; the third group does not respond to any of the compounds tested.

In these sensilla, we have found a new response to divalent cations: In contrast to labellar sensilla in which they are inhibitory, these cations enhance the response of the water cell of leg taste receptors of group I. Furthermore, this response is sexually dimorphic in the pGAL4 Voila strain. We further evaluated if male taste receptors, feminized with a genetical construction (UAS-tra x Voila), expressed the same phenotype.

54

Dolzer Jan, Bittmann Karin and Stengl Monika

Fachbereich Biologie, Tierphysiologie
Philipps-Universität
D-35032 Marburg, FRG

Olfactory adaptation in the hakwmoth Manduca sexta

Key words: Manduca sexta; olfactory receptor neuron; cyclic GMP; adaptation

In response to strong stimulation sensory organs reduce their sensitivity to subsequent stimuli. This process has been termed adaptation. In moth olfactory sensilla adaptation is achieved by reducing the responsiveness of at least two levels in the chemo- electrical transduction machinery. One is the generation of receptor potentials, the other one is their transformation into action potentials. We investigated the mechanisms of adaptation in pheromone-sensitive olfactory receptor neurons (ORNs) of the hawkmoth Manduca sexta using different experimental approaches.

By means of extracellular tip recordings (Kaissling 1974, in: Jaenicke (ed.): Biochemistry of Sensory Functions, Springer) we examined adaptation processes, as well as their time course in individual olfactory sensilla in response to strong adapting stimuli of two different durations. With adapting stimuli of one second the dose-response curves of the adapted sensillar potential (SP) was shifted to the right by at least 1 log-unit: the amplitude of the adapted SP was lower, the rise was slower and the decline was faster. After adapting stimuli of 250 ms, in contrast, the rise time of the adapted SP did not change as it did in response to adapting stimuli of 1 s. Thus, we assume that different mechanisms of adaptation occur in response to different time courses of adapting stimuli.

Biochemical experiments have shown slow rises of cyclic GMP (cGMP) concentrations in response to pheromone stimulation lasting for several seconds (Ziegelberger et al. 1990, J Neurosci 10(4):1217-1225; Boekhoff et al. 1993, Insect Biochem Molec Biol 23(7):757-762), suggesting that cGMP might be involved in one of the mechanisms of adaptation. With NADPH diaphorase histochemistry, cGMP antibodies, and in situ hybridizations (in collaboration with Dr. Alan Nighorn, ARL Tucson, AZ) it was investigated, whether a nitric oxide (NO) synthase or an NO- dependent guanylyl cyclase are present in ORNs. After prolonged exposure to pheromone, a number of pheromone-dependent ORNs was seen to upregulate intracellular cGMP concentrations via an unknown mechanism. A subpopulation of the ORNs was NADPH diaphorase-positive only after pheromone exposure for several minutes, but not for seconds or hours. However, no NO-dependent upregulation of cGMP concentrations was detected in pheromone- dependent ORNs, nor did they contain the alpha- or beta subunits of the soluble, NO-dependent guanylyl cyclase. Therefore, cGMP levels appear to rise in pheromone-dependent cells in an NO- independent manner.

To investigate the physiological effects of cGMP on ion channels possibly involved in the transduction cascade, cell-attached and inside-out patch clamp recordings were performed. At least one type of potassium current, as well as presumptive chloride channels were affected by the application of the membrane- permeant analogue 8-bromo cGMP in different ways. Their further characterization and the elucidation of their physiological relevance are the central questions of our current research.

Supported by DFG STE 531/5-1, 10-1, 10-2

55

Distler P.G.

Institute for Zoology
University of Regensburg
D-93040 Regensburg
Germany
Paul.Distler@biologie.uni-regensburg.de

Characterization of odor-induced activity patterns in the fly antennal lobe by metabolic 2-deoxyglucose labeling

Odor-induced activity patterns have been studied in the central olfactory pathway of the blowfly Calliphora vicina (Diptera) by metabolic [3H] 2-deoxyglucose (2-DG) labeling. High-resolution autoradiography of antennal lobe neuropil reveales that single odorants of different classes (aliphatic alcohols, fatty acids, aromatics and terpenes) elicit a stimulus-specific, spatially distributed activity pattern. These labeling patterns are consistent between individuals. The extend of 2-DG uptake induced by a specific odorant in the antennal lobes increases with higher odor concentration. For a given stimulus, the distribution of 2-DG activity is similar in both brain hemispheres. The majority of 2-DG labeling in the antennal lobe glomeruli is correlated with the central projections of chemoreceptor cell axons. The patterns, therefore, reflect a chemotop map of the sensory antennal input. Despite differences in the molecular structure of the stimuli, the 2-DG uptake is partially overlapping in certain identified glomeruli even at low odor concentrations. The findings suggest that single odorants activate several types of chemoreceptor cells with partly overlapping receptive field and different sites of axonal projections. Considering the known physiological data on chemoreceptor cells in Calliphora, the partially overlapping 2-DG activity patterns (cf. Hexanol/Pinene or Decanol/Pinene) indicate that some glomeruli in the antennal lobe might receive axonal projections from two different types of chemoreceptor cells.

56

Rospars Jean Pierre, Hildebrand John G.

Unite de Biometrie
Institut National de la Recherche Agronomique (INRA)
Versailles, France

ARL Division of Neurobiology,
University of Arizona,
Tucson, AZ 85721, USA

Sexual dimorphism of the glomerular organization in the antennal lobes of the moth Manduca sexta.

Male and female antennal lobes of Manduca sexta were compared with the aim of investigating the nature and extent of the sexual dimorphism of the primary olfactory center of this Lepidopteran species. Complete identification of the glomeruli led to the conclusion that all female gomeruli could be considered as homologous to male glomeruli. This means that no sex-specific glomerulus could be demonstrated in either the male or the female. However, sexual dimorphism was present and has been found to be restricted to the three glomeruli composing the male macroglomerular complex and their female homologs. Only two of these, the large female glomeruli, were previously known. The third component, the male toroid 2, was tentatively identified with a normal sized glomerulus of the female. No other sexual dimorphism was demonstrated among the 60 "ordinary" glomeruli that make up the rest of the glomerular neuropil.

57

Schachtner J.¹ , Menon K.² and Homberg U.¹

¹ FB Biologie, Tierphysiologie, Philipps-University
35032 Marburg
Germany
schachtj@Mailer.Uni-Marburg.DE

² Division of Biology, Caltech
Pasadena, CA 91125
USA

Synaptotagmin immunostaining in the developing antennal lobe of the sphinx moth Manduca sexta

During metamorphosis of the sphinx moth M. sexta, levels of cGMP are transiently upregulated in local neurons of the antennal lobe (AL). This rise in cGMP is partly regulated by NO and correlates in time with the major wave of synapse formation in the AL (Schachtner et al., 1998, J. Comp. Neurol. 396:238-252; Schachtner et al., 1999, J. Neurobiol., in press).

To investigate the possible role of NO and cGMP in synapse formation, we used an antiserum against the ubiquitous synaptic vesicle protein synaptotagmin (syt) for labeling of synaptic contacts in the developing AL. Early in metamorphosis a spheroidal neuropil area stains at the site were the AL develops. Starting about 5 days after pupal ecdysis (P5) the syt staining in the growing AL becomes concentrated in an outer shell area of the neuropil. Beginning on days P7/8 the stained shell area is reorganized into distinctly stained glomeruli. At P11/12, when the main wave of synaptogenesis ends, the glomeruli show an overall robust syt staining. By P16/17 the glomerular staining changes to a more differentiated labeling, suggesting areas of different synaptic density within each glomerulus.

In order to test whether NO and cGMP are involved in synapse formation, we started to pharmacologically interfere with the NO/cGMP signaling pathway. At times when cGMP levels are regulated in vivo, we infused inhibitors of the NO/cGMP pathway. We are currently performing density measurements of the syt immunostaining to detect differences between pharmacologically treated and control animals.

Supported by a DFG grant to J. Schachtner (Scha 678/3-1)

58

Skiri Hanne Therese, Mustaparta Hanna and Almaas Tor Jürgen

Norwegian University of Science and Technology
N-7034 Dragvoll, Norway
hannesk@stud.ntnu.no

Projection of Reseptor Neurons (RNs) in the Macroglomerular Complex (MGC) in the Antennal Lobe (AL) of the moth Helicoverpa armigera.

Electrophysiological recordings from cut hairs of sensilla trichochea type 1 i.e. tip-recordings, were followed by staining of the RNs with cobolt-lysine. In this way the functional types of the RNs and their projections in the MGC were determined. The MGC consist of three major subunits. One large unit ("cumulus") is situated at the entrance of the antennal nerve and two smaller units are located dorso-medially of the larger unit. So far two different types of sensilla are discovered. In the sensillum type containing a reseptor neuron detecting the main pheromone component, Z11-16:Al, two cells were often found. When this sensilla types were stimulated with Z11-16:Al, a single stained axon was seen projecting into "cumulus". In some cases also a second axon was stained, projecting into a smaller glomerulus which probably was an ordinary glumerulus. In a second sensillum type, two reseptor neurons were found and respons to Z9-14:Al were recorded. Weak responses to Z11-16:OH and Z9-16:Al were also found. In this case two axons were stained, which project one to each of the two smaller glomeruli in MGC. It is not yet established wich of the RNs respond to which compound or if it is one RN that respond to all three compounds. In addition the coexistens of the two RNs in the sensillum, makes it difficult to decide which of the stained RNs project to which glomeruli in the MGC. So far no RNs in H. armigera have shown respons to Z11-16:Ac.

Key words: Olfaction - Moth - Pheromone - Single sensillum - Antennal lobe - MGC.

59

Carlsson Mikael and Hansson Bill S.

Department of Ecology, Lund University
S-223 62 LUND, Sweden
mikael.carlsson@ekol.lu.se

Responses in narrowly tuned receptor neurons to binary mixtures of pheromone components and to the complete blend in the turnip moth, Agrotis segetum.

Most electrophysiological studies have used single odorants as stimuli even though mixtures of odorants are generally encountered in nature. The pheromone-detecting receptor neurons in moths are known to be very narrowly tuned but they are not entirely specific. At very high doses they may even respond to host plant odours. This means that the components of a mixture may interact with each other at the receptor sites. A mixture interaction occurs when a response to a blend can not be predicted from the knowledge of the responses to its single components. An interaction may render either a synergistic or a suppressive effect, depending on whether the response is stronger or weaker than predicted. Mixture interactions with food odorants has been shown at the peripheral level e.g. in the spiny lobster (Derby and Ache, 1984) and the Colorado potato beetle (De Jong and Visser, 1988). O'Connell (1985) demonstrated in a single sensillum recording experiment in the moth Trichoplusia ni, that addition of a minor pheromone component enhanced the response to a major pheromone compound in an unpredictable way. Mayer and Doolittle (1995) also found that addition of sex pheromone analogs and minor female produced components synergized the response to the major pheromone component in T. ni.

Interaction between the single components of a pheromone blend is known to take place at the level of the antennal lobe. In the turnip moth, Agrotis segetum, it has been demonstrated that projection neurons and local interneurons may respond to either a single pheromone component and the blend or solely to the blend (Wu et al., 1996). The aim of present study is to investigate coding of mixtures at the receptor neurons in A. segetum. The responses to single components in specialist neurons are compared to responses to binary mixtures of the component the receptor neuron is tuned to and other behaviourly active components. Four different types of neurons tuned to attractive components and one type of neuron tuned to a behaviour antagonist are investigated.

The study demonstrated supression in a few cases, i.e. the observed response to the binary mixture was weaker than would be predicted from the responses to the single components. Synergism was never detected. Strongest supression was shown in the cell tuned to the behaviour antagonist when the second cell, tuned to a major attractant, was spiking simultanously. The mechanisms behind the mixture suppressions are unknown but in the latter case, intercellular interactions can not be excluded.

References
De Jong, R., Visser, J.H. 1988. Specificity-related suppresssion of responses to binary mixtures in olfactory receptors of the Colorado potato beetle. Brain Research. 447: 18-24.
Derby, C.D., Ache, B.W. 1984. Electrophysiological identification of the stimulatory and interactive components of a complex odorant. Chemical Senses 9: 201-218.
Mayer, M.S., Doolittle, R.E. 1995. Synergism of an insect sex pheromone specialist neuron: implications for component identification and receptor interactions. J. Chem. Ecol. 11: 1875-1891.
O`Connell, R.J. 1985. Responses to pheromone blends in insect olfactory receptor neurons. J. Comp. Physiol. 156: 747-761.
Wu, W., Anton, S., Lofstedt, C., Hansson, B.S. 1996. Discrimination among pheromone component blends by interneurons in male antennal lobes of two populations of the turnip moth, Agrotis segetum. Proc. Natl. Acad. Sci. USA. 93: 8022-8027.

60

Chua Gregory C.H. and Kennedy Linda M.

Neuroscience Laboratory
Biology Department
Clark University
Worcester, MA 01601 USA

Feeding on Fructose and Glucose is Greater by Drosophila melanogaster Raised on a Fructose-based Medium than on a Glucose-based Medium

Behavioral and electrophysiological data from Drosophila species and human psychophysical data suggest separate receptor cell mechanisms for fructose and glucose taste (1). The sensitivities for these sugars may involve experience-inducible mechanisms in humans (2, 3). Here we investigated potential inducible mechanisms in D. melanogaster.

Flies were raised in separate media, containing 2% agar, 10% brewer's yeast, 0.6% mold-inhibitor, and fructose 139 mM or glucose 555 mM. Three to eight days after eclosion, flies were tested for their sensitivities to fructose or glucose 8, 16, 32, 64, 128 mM. Groups of flies were food-deprivated (in 1% agar) for 24 hrs in the dark, and then were given a choice between sugars in 1% agar (red) or 1% agar (blue) for 2 hrs (in dark). The numbers of flies having fed were counted according to their abdominal colors.

The percentages (±S.E.) choosing fructose were higher for fructose-raised flies (N= 679) than for glucose-raised flies (N= 859) at all concentrations except 8 and 16 mM, for which they were similar. The percentages (±S.E.) choosing glucose also were higher for fructose-raised flies (N= 580) than glucose-raised flies (N= 851) at all concentrations except 8 and 128 mM, for which they were similar. The percentages choosing agar was £ 0.007% for any sugar concentration, but control tests showed that flies did not prefer red over blue solutions.

These data support the proposal of experience-induced changes in sugar sensitivities, and indicate that experience with fructose leads to greater sensitivities than experience with glucose. Electrophysiological studies of receptor cell firing in response to fructose and glucose by flies in the two treatment groups are in progress to determine whether the experience-induced differences occur in the receptor cells.

Supported by NIH DC/OD02663. To LMK

References:
Kennedy, L.M. et al., Food Chem. 60: 311, 1997
Eylam, S. & L.M. Kennedy, Olfaction & Taste XII, Ann. N.Y. Acad. Sci. 855: 170-174, 1998
Eylam, S. & L.M. Kennedy, Chem. Senses 23: 588. 1998

61

Noorman Nico and Den Otter Cornelis J.

Group Sensory Organs and Behaviour
Department of Animal Physiology
University of Groningen
P.O. Box 14, 9750 AA Haren, The Netherlands
noormann@biol.rug.nl

Variations in muscalure production between and within different strains of houseflies.

Keywords: Musca domestica; sex pheromone; (Z)-9- tricosene; cuticular hydrocarbons;

Summary
Muscalure, (Z)-9-tricosene, is thought to be the major component of the contact sex pheromone of the female house fly Musca domestica.

We investigated the variation in muscalure production by females of a WHO laboratory strain and four wild type strains. The results show that muscalure production in the wild type strains was much lower than in the laboratory strain. In the WHO strain muscalure production was positively related to the production of almost all other cuticular hydrocarbons, while in the wild type strains only a strong relationship existed between (Z)-9-tricosene and tricosane. In wild type strains there was no significant relation between bodyweight and muscalure and tricosane amounts while there was a significant relation between bodyweight and total hydrocarbons present on the skin of females. In the laboratory strain this relationship existed for both muscalure and tricosane on one hand and for the total amounts of hydrocarbons on the other hand.

The results suggest that the production of muscalure and tricosane follow a biological pathway different from the production of other hydrocarbons and that tricosane is a product produced in the same process as muscalure.

62

Sass Hinrich and Pappenberger Barbara

Zoologisches Institut der Universitaet Regensburg
hinrich.sass@biologie.uni-regensburg.de

Responses of antennal olfactory receptors in yellow fever mosquitoes to human body odour and its components

Female Aedes aegyptii are easily attracted in a wind tunnel by odours emanating from a human hand. Ethanol extracts obtained by rubbing the skin with cotton pads, contain essential components for attraction and host location. This extract is the starting material for the identification of attractive components of human body odours. Preparative HPLC and GC is applied to isolate these components. The attractivity of different LC-fractions is tested in a windtunnel as well their ability to stimulate receptor neurons in female mosquito olfactory receptor cells.

A mixture of three LC-fractions reaches the attractive effect of the extract itself. Several receptor cells in A3-type sensilla respond to the extract just as to the native body odour. Responses of cells in other sensillum types have not been found. The most polar fraction contains lactic acid, which is known to be essential for the extract's effectiveness. Two less polar fractions contain components which are also necessary for the attractive effect. Each of them elicits responses in different olfactory receptor neurons. The chemical nature of this compounds has not yet been determined. Ammonia is a component of one of the fractions just as of human body odour. Some receptor cells respond to ammonia. In the windtunnel ammonia increases the attractivity of lactic acid.

Preparative GC in combination with simultanous electrophysiologial registration offers a chance to detect components effective for receptor neurons. These should be candidates for attractive compounds of human body odour.

Fifty GC runs have already been performed. The results confirm, that the extract contains many compounds which receptor neurons respond to. Apparently the specific host odour for mosquitoes consists of many odour compounds. Therefore one has to deal with the analysis of many chemical components. In a first step those regions of the chromatograms are localized, in which receptor cell responses frequently occur.

Although the method has acquired a certain routine, the properties of the preparation itself, as there are a high background activity of the cells and effects, which point to mutual influences between receptor cells in a sensillum present considerable difficulties in spotting significant receptor-cell-responses. In addition different odour compounds of different effectiveness require tests using different amounts of the same starting material.

63

Kroeber T. and Guerin P.M.

Dep. of Animal Physiology
Institute of Zoology
University of Neuchatel
Rue Emile Argand 11
CH-2007 Neuchatel, Switzerland
thomas.kroeber@zool.unine.ch

Ixodid ticks avoid contact with liquid water

Larva of the cattle tick Boophilus microplus and all life stages of the European sheep tick Ixodes ricinus avoid walking on a wet surface surrounding a dry patch.

In 170 reactions made at a border with a film of liquid water on a semipermeable membrane by 22 B. microplus larvae, 40% consisted of immediate turns to the opposite side to bring all legs back onto a dry patch, 41% were walks along the border during which the ticks maintained contact with both the dry and wet zones, and 19% were returns to the dry patch after a short excursion onto the wet surround.

Sequential analysis of the border behaviours showed that repetitive contact with the water at the border of the dry patch confines increased the probability of walks astride the border.

Since contact with one front leg tip was sufficient to cause return reactions from the wet surface in most of the border contacts, the water receptor(s) which permit ticks to perceive the wet surface are probably located in terminal pore sensilla on the first-leg tarsi.

Water avoidance may be of survival value for ticks. Tick larvae turn instantaneously upon touching a water droplet with their front leg tarsi, a behaviour that may be of importance to prevent confinement in such drops from which the tick larvae cannot escape due to the high surface tension of the water. Ticks are found in nature on hydrophobic surfaces such as the cuticle of congeners when aggregating, the sebum covered pelage of hosts, and the waxy cuticle of plants which act as ambush sites. Avoiding contact with ground water in which pathogens circulate helps ticks to avoid infection.

64

Oppliger F.Y., Guerin P.M. and Vlimant M.

Institut fuer Zoologie
Université de Neuchâtel
CH-2007 Neuchâtel, Schweiz
patrick.guerin@zool.unine.ch

Neurophysiological and behavioural evidence for an olfactory function for the dorsal organ and a gustatory one for the terminal organ in Drosophila melanogaster larvae

Multicellular electrophysiological responses from the dorsal organ on the cephalic lobes of third instar Drosophila melanogaster larvae (wild-type Canton S) stimulated with a cold-trapped banana volatile extract showed that this structure has an olfactory function in the fruit fly. Responses of the dorsal organ were also recorded to constituents of the banana volatile extract as they eluted from a gas chromatographic column (GC-coupled dorsal organ electrophysiology). The active chemostimulants were identified as 2-heptanone, isoamyl alcohol, hexyl acetate, hexanol and hexyl butyrate by gas chromatography-coupled mass spectrometry. Applying the same recording system to the terminal organ sensilla, no responses were obtained to either the banana volatile bouquet or its constituents. By contrast, high frequency multicellular responses were recorded in response to touching the terminal organ with the gustatory stimuli KCl and grapefruit juice; responses were absent in similar stimulation of the dorsal organ with either NaCl or KCl. This suggests a role for olfaction by the dorsal organ and for gustation by the terminal organ in Drosophila larvae.

In a 7mm high wind tunnel with a thin 1.2% agar floor, the Drosophila larvae showed odour-conditioned anemotactic responses in an air stream of 0.1 m/sec bearing banana volatiles. Drosophila larvae responded best to the odour of cut bananas. A 1:1 mixture of the banana odour constituents 2-heptanone and hexanol (at either 50 µg or 100 µg source dose each) proved as attractive as the known larval attractants proprionic acid and isoamyl acetate on their own at 100mg, whereas hexanol and 2-heptanone on their own at a 100 µg source dose were less attractive. The stronger behavioural response to the banana volatile bouquet and to the binary mixture serves to underline the multireceptor nature of the dorsal organ response to food odour in Drosophila.

65

INOUE A. Takashi¹ , OZAKI Mamiko² , ASAOKA Kiyoshi¹ , SETA Kazuaki³

¹ Japanese National institute of
Sericultural and Entomological Science,
1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan
inoueatp@ss.nises.affrc.go.jp

² Biopark of Adachi, Tokyo
Hogima, Adachi-ku,
Tokyo 121- 0064, Japan

³ Kyoto Institute of Technology
Goshokaido-cho, Matsugasaki,
Sakyo-ku, Kyoto 606-8585, Japan

Sensory basis for the ovipositional behavior in the swallowtail butterfly, Papilio protenor: Eletrophysiological responses of the female tarsal chemosensillum to several compounds in the Rutaseae plants.

According to the behavioral experiments with the swallowtail butterfly, Papilio protenor, by Honda et al. (1986, 1988, 1990), several compounds isolated from its host and non-host Rutaseae plants were identified as stimulus and inhibitory substances for their oviposition. We examined the electrophysiological responses to these compounds in the foreleg tarsal chemosensilla of the female butterflies. As the results, these compounds were classified into 4 groups as follows,
Group 1.    Phellamurin (flavonoid : inhibitory), Caffeic acid (phenylpropanoid : no-effects?); Long and short types of impulses reproductively appeared.
Group 2.    Naringin (flavonoid : stimulus), Chlorogenic acid (phenylpropanoid : stimulus), Short type of impulses reproductively appeared.
Group 3.    Hesperidin (flavonoid : stimulus), Rutin (flavonoid : no-effect), Quinic acid (sugar acid : stimulus), Some times, short type of impulses appeared.
Group 4.    Proline (amino acid : stimulus), Synephrine (tyramine-like, stimulus); Few impulses appeared.
We considered that the long and the short types of impulses might be sensory signals to inhibit and stimulate ovipositional behavior of the butterfly, respectively.

66

Ziesmann Jürgen and Maida Rosario

Max-Planck-Institut für Verhaltensphysiologie Seewiesen
Postfach 1564, 82305 Starnberg

Comparison of olfactory physiology and biochemistry of the Saturniid moths Antheraea polyphemus and Attacus atlas

One of the important unanswered questions about insect odorant binding proteins is: how specifically do they select the odorant to bind? We investigated this by comparing the physiology and biochemistry of two Saturniid moths. Antheraea polyphemus has two main sex pheromone components: (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-6,11-hexadecadienal (Kochansky et al. 1975, J. Insect Physiol. 21, 1977-1983 ). The pheromone of the second species, Attacus atlas, is not yet known, thus in our experiments we tested pentane and acetone extracts of the sex-pheromone glands of the females.

EAG experiments: As expected, the males of each species give a large EAG response to the pheromones of their own females but do not respond to the pheromone of the other species. Detection of their own pheromone is rarely found in Lepidoptera and also the females of both Attacus and Antheraea do not respond to their own pheromone. The females of A. polyphemus also do not respond to Attacus gland extracts, but only to some flower and green leaf volatiles. Surprisingly, Attacus females do respond to both pheromone components of Antheraea with a clear EAG in a dose dependent manner.

Biochemistry: In both species and sexes we find General Odorant Binding Proteins, GOBP1 and GOBP2. Females contained larger amounts of the two GOBPs than males. Additionally, in both species males have Pheromone Binding Protein (PBP) in their antenna, while PBP in females is only faintly detectable. Aldehyde oxidase and sensillar esterase, two enzymes involved in pheromone degradation were found in males antennae of A. polyphemus but not in female antennae. The opposite situation is found in A. atlas (see table). In spite of the fact that the A. atlas males respond to their female gland extract, i.e. to the unknown pheromone, we could not detect any of the above pheromone degrading enzymes. Instead, female Attacus possess both, the sensillar esterase and the aldehyde oxidase (see table).

	EAG to A. polyphemus pheromone	EAG to A. atlas pheromone	sensillar esterase	aldehyde oxidase	PBP	GOBP1	GOBP2
A. poly. females	0	0	0	0	0	++	++
A. poly. males	++	0	++	++	++	+	+
A. atlas females	++	0	++	++	0	++	++
A. atlas males	0	++	0	0	++	+	+

Conclusions: Attacus atlas males respond to female gland extracts and contain PBP, but surprisingly no sensillar esterase and aldehyde oxidase as pheromone degrading enzymes. This suggests that the pheromone of A. atlas is not a C-16 hexadecadyenyl acetate nor a C-16 hexadecadyenal. Female Attacus atlas respond to A. polyphemus pheromones and possess the enzymes necessary for their degradation. Therefore A. atlas females resemble the males of A. polyphemus in this respect and seem adapted to detect molecules with a very similar structure to the pheromones of A. polyphemus. As there is only very little PBP found in female A. atlas the pheromone molecules are probably bound to one of the GOBPs of the females. There is no geographical overlap reported for the two species but there may be other species with similar pheromones in this geographical range. Thus, the biological importance of the ability of the females to detect the pheromones of a different species remains unknown.

67

van der Pers Jan N.C.

SYNTECH / VDP Laboratories
P.O.Box 1547
NL-1200 BM Hilversum
syntech@knoware.nl

A multichannel data acquisition system for the USB

A four channel data acquisition system developed for interfacing with a personal computer (PC) via the recently introduced Universal Serial Bus (USB) is presented and demonstrated. The system can be connected to any PC supported with an USB port; no interface board needs to be installed in the PC. Data acquisition via the USB has the advantage that no dedicated PC is needed for signal recording, and that also portable computers with an USB port can be used.

The system consists of a compact unit containing the signal input receptacles, amplifiers, A/D converters, and logic circuits for the USB data transfer. Furthermore, a power supply is built-in to energize active input devices like high impedance headstages. No separate amplifiers or power supplies are needed. Gain, offset, 50 or 60 Hz notch filter, and low and high pass filter characteristics of the four independent amplifiers are software controlled, and the settings are stored with the recorded data. The sampling rate can be selected from 1/s to 45k/s for each channel individually. In addition to the four analog signal inputs up to 8 digital (on/off) inputs are available for recording of event-related and trigger signals. All inputs and power lines are galvanically isolated to eliminate the risk of ground loops in the external wiring.

The software runs under Windows 98 and the coming version of Windows NT (2000) and is written in 32 bits code for efficient operation. Before and during recording all signals are displayed in real time mode on the PC screen, and the time base speed can be adjusted like on an oscilloscope. Multiple recordings are arranged and stored together in a project file rather than in separate files, and all amplifier and software settings are automatically stored with the data.

The stored signals can be processed and analysed by a large variety of software tools. Operations are possible on single recordings as well on groups; All signals stored in a project file are accessible and can be displayed on a common time base. Spikes are identified, extracted from the analog waveform, and classified according to user-defined parameters; Spike frequency in any section of a recording is interactively measured; DC fluctuations are validated using cursor operated interactions; Signal averaging on internal or external events, and auto- and cross colleration functions are available.

An interesting option is the possibililty to replay the stored analog signals via the sound card in the PC. This option allows the replay of voice comments recorded on one of the channels during acquisition of the signals.

The software is continuously upgraded with additional tools and implementations of suggestions and wishes from users.

68

Oldenburg Corina

Max-Planck-Institut füer Verhaltensphysiologie Seewiesen,
82319 Starnberg, Germany

Degradation of synthetic pheromones by environmental factors: laboratory and field experiments

The synthetic pheromone of the European grape moth Lobesia botrana was heated to 100^oC and exposed to UV light (302 nm). It was examined before and after the degradation by means of a GC-MS(D), and tested for its attractivity by field trapping. The degradation products of the irradiated samples did not attract Lobesia botrana. Some of the traps did attract insects belonging to other species (Cydia pomonella, Lymantria dispar). The structure of several degradation products was discussed. It turned out that oxydative together with thermal processes are involved in the observed pheromone degradation. Unlike the pheromone of Lobesia botrana with conjugated double bonds, the synthetic pheromone components of three other moth species without conjugated double bonds showed under the same conditions a good stability.

69

Muroni Patrizia, Tomassini Barbarossa Iole & Angioy Anna Maria

Department of Experimental Biology - Section of General Physiology
University of Cagliari, 09042 Monserrato - Cagliari, Italy
amheart@unica.it

Cardiac responses to mechanical stimulation and to odorants in blowfly pupae

The insect heart represents an experimental material with unique biological properties and evolutionary conserved characteristics, having close ultrastructural and functional affinities with the mammalian myocardium. As concerns neural control mechanisms of heart activity, cardiac responses to external sensory stimulation have been described in a variety of insect species, in analogy with responses of vegetative type tipical of higher animal species. For instance, olfactory stimulation with sex pheromone or hexanol vapors induces a clear cut tachicardiac response in Heliothis virescens and Spodoptera littoralis adult moths. Stimulation of taste or olfactory receptors elicits an immediate arrest of forward beating and a sudden setting in of backward beating of the heart in adult blowflies. On the basis of low threshold and short latency values, cardiac responses are described as a sensitive, reliable tool for testing the reactivity of adult insects to chemical stimuli.

In the present research study we have evaluated if cardiac responses could occur during the pupal developmental stage of the insect as well. To this end, we studied regular heart activity, i.e. in the absence of stimulation, and its possible variations following mechanical or olfactory stimulation. Experiments were performed on Protophormia terraenovae flies, at the beginning (24-hours-old; n = 66) and at the end (24 hours before the emergency; n = 53) of the pupal stage. Heart activity before and following stimulation was continuously monitored on intact specimens by recording electrocardiograms with metal electrodes positioned on the puparium or on the innermost pupal cuticle. Two modes of mechanical stimulation were used. A first one consisted of an intense, charcoal-filtered and humidified air stream (1.5 l/min) that was delivered through a glass tube ending 20 mm in front of the pupa (main air stream). Stimulation lasted for 1 s. As a second mode, stimulation was performed by lightly touching the cuticular area with the tip of a pin using a micromanipulator. Odorants were delivered over the pupa by means of an olfactometer. The specimen was continuously flushed with an intense air stream, which was identical to the one used for performing mechanical stimulation. A secondary air stream (0.1 l/min) of clean air (control), or air containing vapors of the stimulant chemical(ammonia, hexanol) at different concentrations, was added for 1 s to the main one.

In younger pupae, the cardiac cycle before stimulation consisted of a regular alternance of a spike activity period with an isoelectric period. In all tested specimens, an immediate and transient arrest of spike activity was detected following tactile stimulation. Cardiac arrest occurrence vanished as a function of repetitive stimulation and a period of several minutes was required for a recovery of cardiac reactivity. Any other kind of stimulation was ineffective, even though a 28% decrease in the duration of the spike activity period was measured following the delivery of ammonia vapors in 25% of specimens.

In older pupae, the cardiac cycle before stimulation consisted of a regular alternance of two different phases of activity occurring at a higher (fast phase) and a lower (slow phase) spike frequency. A constant pattern of changes in regular activity, consisting of an immediate arrest of the fast phase and a sudden setting in of a slow phase, followed mechanical and olfactory stimulation in 90% of specimens. The response could occur several times in sequence following repetitive odor delivery. Cardiac reactivity was always recovered by allowing a short time period in absence of stimulation.

The results of this investigation show that cardiac responses are effective indicators of mechanical or olfactory reception in fly pupae. They therefore might be considered as a useful tool for testing the biological importance of environmental factors in developmental studies.

Funding: Italian Ministry of University and Scientific Research

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